Mutant 3-hydroxybutyrate dehydrogenase from Rhodobacter sphaeroides as well as methods and uses involving the same

ABSTRACT

The present invention relates to a mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) with improved performance relative to the wild-type 3-HBDH, a nucleic acid encoding the mutant 3-HBDH, a cell comprising the mutant 3-HBDH or the nucleic acid, a method of determining the amount or concentration of 3-hydroxybutyrate in a sample, and a device for determining the amount or concentration of 3-hydroxybutyrate in a sample.

The present invention relates to a mutant 3-hydroxybutyratedehydrogenase (3-HBDH) with improved performance relative to thewild-type 3-HBDH, a nucleic acid encoding the mutant 3-HBDH, a cellcomprising the mutant 3-HBDH or the nucleic acid, a method ofdetermining the amount or concentration of 3-hydroxybutyrate in a sampleand a device for determining the amount or concentration of3-hydroxybutyrate in a sample.

Ketone bodies are produced in the liver, mainly from the oxidation offatty acids, and are exported to peripheral tissues for use as an energysource. They are particularly important for the brain, which has noother substantial non-glucose-derived energy source. The two main ketonebodies are 3-hydroxybutyrate and acetoacetate. Biochemically,abnormalities of ketone body metabolism can be subdivided into threecategories: ketosis, hypoketotic hypoglycemia, and abnormalities of the3-hydroxybutyrate/acetoacetate ratio.

An abnormal elevation of the 3-hydroxybutyrate/acetoacetate ratiousually implies a non-oxidized state of the hepatocyte mitochondrialmatrix resulting from hypoxia-ischemia or other causes.

The presence of ketosis normally implies that lipid energy metabolismhas been activated and that the entire pathway of lipid degradation isintact. In rare cases, ketosis reflects an inability to utilize ketonebodies. Ketosis is normal during fasting, after prolonged exercise, andwhen a high-fat diet is consumed. During the neonatal period, infancyand pregnancy, times at which lipid energy metabolism is particularlyactive, ketosis develops readily.

Pathologic causes of ketosis include diabetes, ketotic hypoglycemia ofchildhood, corticosteroid or growth hormone deficiency, intoxicationwith alcohol or salicylates, and several inborn errors of metabolism.

The formation of ketone bodies is increased when lipolysis is increasede.g. in insulin deficiency (diabetes mellitus; in particular type Idiabetics), when the glucagon concentration is increased and in afasting state. In such cases the normal physiological concentration ofless than 7 mg/dl can increase to more than 10-fold. Over the past years3-hydroxybutyrate has proven to be an extremely reliable parameter formonitoring an insulin therapy.

The absence of ketosis in a patient with hypoglycemia is abnormal andsuggests the diagnosis of either hyperinsulinism or an inborn error offat energy metabolism.

Accordingly, ketone bodies are an interesting diagnostic target,particularly for diabetes. Therefore, there is an ongoing need forrobust and sensitive test-systems for ketone bodies, especially3-hydroxybutyrate. The ratio of 3-hydroxybutyrate to acetone oracetoacetic acid is normally 3:1. In keto-acidoses the ratio increasesto 6:1 to 12:1. A suitable 3-HBDH should enable the development of aquantitative test for 3-hydroxybutyrate.

Surprisingly, it has been found that a variety of mutants of the3-hydroxybutyrate dehydrogenase from Rhodobacter sphaeroides showimproved performance, particularly increased thermal stability and/oraffinity for substrate and/or cofactor. As shown in the Examples, thereare many sites at the wild-type enzyme which allow for mutations, whichincrease thermal stability and/or affinity for substrate and/or cofactorof the mutant relative to the wild-type enzyme.

The inventors identified a considerable amount of amino acids in thewild-type 3-HBDH, the substitutions of which increased the performanceof the resulting mutants relative to the wild-type HBDH (see e.g. Table1A and 1B of the Examples). Particularly, amino acids may be substitutedto increase thermal stability and optionally affinity for substrateand/or cofactor (for 3-hydroxybutyrate and/or NAD or derivatives of NAD,e.g. carba-NAD). Suitable examples of those sites include positions 66,109 113, 125, 140, 144, 145, 195, 197, 217, 223, 232, 239, 250 and/or257 of SEQ ID NO: 1. The performance of the mutant 3-HBDH could befurther increased by combining mutations at the above or further sites(see Tables 2A and 2B). Particularly suitable examples of those mutantsinclude those with the mutations (for definitions of abbreviations seebelow)

-   -   125Phe;    -   144Arg;    -   232Cys;    -   250Met;    -   230Leu and 250Met;    -   37Tyr and 250Met;    -   37Tyr, 230Leu and 250Met;    -   202Gly, 230Leu and 250Met;    -   37Tyr, 145Gly, 230Leu and 250Met; or    -   37Tyr, 195Gln, 230Leu and 250Met        (see Examples).

Accordingly, in a first aspect, the present invention relates to amutant 3-hydroxybutyrate dehydrogenase (3-HBDH) from Rhodobactersphaeroides with improved performance relative to the wild-type 3-HBDH.Preferably, the mutant 3-3-HBDH from Rhodobacter sphaeroides withimproved performance relative to the wild-type 3-HBDH comprises an aminoacid sequence that is at least 80% identical to the amino acid sequenceof SEQ ID NO: 1 (3-HBDH from Rhodobacter sphaeroides; wild-type 3-HBDH)and has at least one amino acid substitution relative to the wild-type3-HBDH at a position corresponding to position 250 of SEQ ID NO: 1. Morepreferably, the amino acid at the position corresponding to position 250of SEQ ID NO: 1 is substituted with Met (250Met) or Ile (250Ile),particularly with Met (250Met). The position and substitutions have beenproven to be advantageous in order to improve performance of a mutant3-HBDH (see Tables 1A and 1B) and may be combined with further mutations(see Tables 2A and 2B). Accordingly, particularly suitable mutantsinclude those with the mutations

-   -   250Met;    -   230Leu and 250Met;    -   37Tyr and 250Met;    -   37Tyr, 230Leu and 250Met;    -   202Gly, 230Leu and 250Met;    -   37Tyr, 145Gly, 230Leu and 250Met; or    -   37Tyr, 195Gln, 230Leu and 250Met        (see Examples).

3-Hydroxybutyrate dehydrogenase (3-HBDH) (EC 1.1.1.30) belongs to thefamily of oxidoreductases, to be specific is an enzyme that catalyzesthe stereospecific oxidation of3-hydroxybutanoate/3-hydroxybutyrate/(R)-3-hydroxybutyrate/3-hydroxybutyricacid (3-HB) to acetoacetate with NAD or derivatives as cofactor:(R)-3-hydroxybutyrate+NAD⁺

acetoacetate+NADH+H⁺

The systematic name of this enzyme class is (R)-3-hydroxybutanoate:NAD⁺oxidoreductase. Other names in common use includeNAD⁺-beta-hydroxybutyrate dehydrogenase, hydroxybutyrate oxidoreductase,beta-hydroxybutyrate dehydrogenase, D-beta-hydroxybutyratedehydrogenase, D-3-hydroxybutyrate dehydrogenase,D-(−)-3-hydroxybutyrate dehydrogenase, beta-hydroxybutyric aciddehydrogenase, 3-D-hydroxybutyrate dehydrogenase, andbeta-hydroxybutyric dehydrogenase. This enzyme participates in synthesisand degradation of ketone bodies and butanoate metabolism and it may beused in order to determine ketone bodies in samples, such as a bloodsample.

The term “wild-type 3-HBDH” relates to a 3-HBDH as it typically occursin nature. A wild-type 3-HBDH from the purple photosynthetic bacteriumRhodobacter sphaeroides was cloned in E. coli and further described in1999 (DE 19815685 A1). However, the enzyme is characterized by a ratherlow stability (resulting in short storage life), which isdisadvantageous in biotechnical, biomedical and diagnostic applications.Particularly, the short storage life has a negative impact onapplicability of the enzyme, if the use of a previously prepared enzymeis intended. This includes particularly commercial products such asenzyme preparations, kits, test strips etc, which are usually preparedin large scale, stored and then marketed e.g. in smaller batches.Additionally, it is evidently desirable to increase the enzyme'saffinity for the substrates and/or cofactors. Increased affinity and/orstability will increase the performance of the enzyme in biotechnicalapplications and devices. The commonly accepted amino acid sequence ofthe typically occurring wild-type 3-HBDH of Rhodobacter sphaeroides isgiven below as SEQ ID NO:1.

The term “mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) fromRhodobacter sphaeroides” relates to a 3-HBDH enzyme whose amino acidsequence differs from the wild-type 3-HBDH from Rhodobacter sphaeroides,particularly the amino acid sequence of SEQ ID NO:1, by at least onemutation, i.e. one or more amino acid substitutions, additions,deletions or combinations thereof, particularly by at least onesubstitution.

The term “with improved performance relative to the wild-type 3-HBDH”means that the performance of the mutant enzyme is improved relative tothe wild-type 3-HBDH from Rhodobacter sphaeroides. The performance isimproved, if the mutant enzyme has a higher performance, e.g. higheractivity in converting 3-HB into acetoacetate, at any condition (e.g.after storage, at a particular pH, with a specific buffer, at a chosentemperature, with a particular cofactor (e.g. carba-NAD), at a specificsubstrate or cofactor concentration etc). Higher performance may beespecially increased stability (such as thermal stability) and/oraffinity for one or more substrates/cofactors (e.g. carba-NAD and/or3-HB). Preferably, the performance is improved, if the mutant enzyme,e.g., has a higher relative remaining activity in converting 3-HB intoacetoacetate, upon being stressed with given conditions (e.g. storage,buffer conditions, temperature, concentration of cofactor or substrate)compared to the activity without being stressed under these conditionsand/or if the mutant enzyme, e.g., has a higher relative activity (i.e.activity at a subsaturation concentration/activity at a saturationconcentration).

The sequence of the wild-type 3-HBDH from Rhodobacter sphaeroides isshown as SEQ ID NO: 1:

(3-HBDH from Rhodobacter sphaeroides) SEQ ID NO: 1MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTISV DGGWTAL 257

Accordingly, a preferred example of a naturally occurring wild-type3-HBDH is given above as SEQ ID NO: 1.

If one or more mutations are introduced into the wild-type sequence,especially the sequence of SEQ ID NO: 1, a mutant 3-HBDH is obtained.With respect to the mutant 3-HBDH of the present invention it is notedthat the mutant is functionally active and shows improved performancerelative to the wild-type 3-HBDH. This means that the mutant hasmaintained its enzymatic function of conversion of 3-HB intoacetoacetate. Additionally, preferably at least the stability andoptionally the enzyme's affinity for the substrate and/or cofactors isincreased. The mutant differs from the wild-type 3-HBDH by one or moreamino acid substitutions, additions and/or deletions, especially atleast one or more substitutions.

The sequence of the mutant 3-HBDH according to the present inventioncomprises (optionally in addition to the substitutions specified herein)one or more amino acid substitution(s), deletion(s) or addition(s),especially one or more substitutions, particularly one or moreconservative amino acid substitutions.

In one embodiment of the present invention, the mutant 3-HBDH accordingto the present invention may comprise one or more amino acidsubstitution(s), particularly a limited number of substitutions (e.g. upto 50, 40, 30, 20 especially 10 amino acid substitutions). Suitablesubstitutions are given in the Examples, particularly in the Tables. Allthe substitutions identified in the Examples as suitable in order toincrease performance of 3-HBDH may be used in the invention either aloneor in combination with each other and/or with further substitutions,e.g. conservative substitutions. “Conservative amino acid substitution”refers to a substitution of a residue with a different residue having asimilar side chain, and thus typically involves substitution of theamino acid in the polypeptide with amino acids within the same orsimilar defined class of amino acids. By way of example and notlimitation, an amino acid with an aliphatic side chain may besubstituted with another aliphatic amino acid, e.g., alanine, valine,leucine, and isoleucine; an amino acid with hydroxyl side chain issubstituted with another amino acid with a hydroxyl side chain, e.g.,serine and threonine; an amino acid having aromatic side chains issubstituted with another amino acid having an aromatic side chain, e.g.,phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with abasic side chain is substituted with another amino acid with a basicside chain, e.g., lysine and arginine; an amino acid with an acidic sidechain is substituted with another amino acid with an acidic side chain,e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Examples of conservative amino acid substitutionsinclude those listed below:

Original Residue Conservative Substitutions Ala, Leu, Val, Ile Otheraliphatic (Ala, Leu, Val, Ile) Other non-polar (Ala, Leu, Val, Ile, Gly,Met) Gly, Met Other non-polar (Ala, Leu, Val, Ile, Gly, Met) Asp, GluOther acidic (Asp, Glu) Lys, Arg Other basic (Lys, Arg) Asn, Gln, Ser,Thr Other polar (Asn, Gln, Ser, Thr) His, Tyr, Trp, Phe Other aromatic(His, Tyr, Trp, Phe) Cys, Pro None

In one embodiment of the present invention, the mutant 3-HBDH accordingto the present invention may comprise one or more amino acidaddition(s), particularly small (e.g. up to 50, 40, 30, 20 especially 10amino acids) internal amino acid additions. Alternatively, additions maybe achieved by combining the mutant 3-HBDH into a fusion proteincomprising the mutant 3-HBDH of the present invention.

Fusion proteins are proteins created by joining of two or moreoriginally separate proteins or peptides. This procedure results in apolypeptide with functional properties derived from each of the originalproteins. Accordingly, depending on the intended use of the 3-HBDH itmay be combined with a further peptide or protein into a fusion protein.The proteins may be fused via a linker or spacer, which increases thelikelihood that that the proteins fold independently and behave asexpected. Especially in the case where the linkers enable proteinpurification, linkers in protein or peptide fusions are sometimesengineered with cleavage sites for proteases or chemical agents thatenable the liberation of the two separate proteins. Di- or multimericfusion proteins can be manufactured through genetic engineering byfusion to the original proteins of peptide domains that induceartificial protein di- or multimerization (e.g., streptavidin or leucinezippers). Fusion proteins can also be manufactured with toxins orantibodies attached to them. Other fusions include the addition theaddition of signal sequences, such a lipidation signal, sequence, asecretion signal sequence, a glycosylation signal sequence, atranslocation signal peptide etc.

Preferably, the fusion protein of the present invention comprises a tag.Tags are attached to proteins for various purposes, e.g. in order toease purification, to assist in the proper folding in proteins, toprevent precipitation of the protein, to alter chromatographicproperties, to modify the protein or to mark or label the protein.Examples of tags include Arg-tag, the His-tag, the Strep-tag, theFlag-tag, the T7-tag, the V5-peptide-tag, the GST-tag and the c-Myc-tag.

In one embodiment of the present invention, the mutant 3-HBDH accordingto the present invention may comprise one or more amino aciddeletion(s), particularly N- and/or C-terminal deletions. The deletionsmay be small (e.g. up to 50, 40, 30, 20, especially 10 amino acids).

In another embodiment, the sequence of the mutant 3-HBDH according tothe present invention may comprise, preferably in addition to thesubstitutions specified herein, a combination of one or moredeletion(s), substitution(s) or addition(s) as defined above.

In a preferred embodiment of the present invention, the mutant 3-HBDHcomprises an amino acid sequence that is at least 80% identical to theamino acid sequence of SEQ ID NO: 1 (3-HBDH from Rhodobactersphaeroides; wild-type 3-HBDH).

The term “at least 80% identical” or “at least 80% sequence identity” asused herein means that the sequence of the mutant 3-HBDH according tothe present invention has an amino acid sequence characterized in that,within a stretch of 100 amino acids, at least 80 amino acids residuesare identical to the sequence of the corresponding wild-type sequence.Sequence identities of other percentages are defined accordingly.

Sequence identity according to the present invention can, e.g., bedetermined by methods of sequence alignment in form of sequencecomparison. Methods of sequence alignment are well known in the art andinclude various programs and alignment algorithms which have beendescribed in, e.g., Pearson and Lipman (1988). Moreover, the NCBI BasicLocal Alignment Search Tool (BLAST) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.Percentage of identity of mutants according to the present inventionrelative to the amino acid sequence of e.g. SEQ ID NO: 1 is typicallycharacterized using the NCBI Blast blastp with standard settings.Alternatively, sequence identity may be determined using the softwareGENEious with standard settings. Alignment results can be, e.g., derivedfrom the Software Geneious (version R8), using the global alignmentprotocol with free end gaps as alignment type, and Blosum62 as a costmatrix.

As detailed above, the mutant 3-HBDH of the present invention in oneembodiment comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 1. In a preferredembodiment, the mutant 3-HBDH comprises or consists of an amino acidsequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to any of the amino acid sequences of SEQ ID NO: 1. Sequenceidentity may be determined as described above.

In another preferred embodiment the mutant of the present invention hasat least one amino acid substitution relative to the wild-type 3-HBDH,wherein the at least one amino acid substitution is at one or more ofthe position(s) corresponding to position(s) 66, 109, 113, 125, 140,144, 145, 195, 197, 217, 223, 232, 239, 250 and/or 257 of SEQ ID NO: 1,particularly at least position(s) 125, 144, 232 and/or 250 of SEQ ID NO:1, more particularly at least position(s) 125, 144, and/or 250 of SEQ IDNO: 1, especially at least position 250 of SEQ ID NO: 1. It has beenshown in the Examples that substitution of the amino acid at theposition corresponding to the above positions of SEQ ID NO: 1 increasesstability and/or affinity for the substrates and/or cofactors (see e.g.Tables 1A and 1B). Substitutions at these positions may be combined witheach other (see also Examples).

Preferably, the mutant 3-3-HBDH from Rhodobacter sphaeroides withimproved performance relative to the wild-type 3-HBDH comprises an aminoacid sequence that is at least 80% identical to the amino acid sequenceof SEQ ID NO: 1 (3-HBDH from Rhodobacter sphaeroides; wild-type 3-HBDH),has at least one amino acid substitution relative to the wild-type3-HBDH at a position corresponding to position 250, preferably 250Met,of SEQ ID NO: 1 and has at least one further amino acid substitution atone or more of the position(s) corresponding to position(s) 66, 109 113,125, 140, 144, 145, 195, 197, 217, 223, 232, 239 and/or 257 of SEQ IDNO: 1, particularly at least position(s) 125, 144 and/or 232 of SEQ IDNO: 1, more particularly at least position(s) 125 and/or 144, of SEQ IDNO: 1.

Particularly suitable examples of substitutions for a mutant 3-HBDH ofthe present invention are given in the following:

-   -   position 66 of SEQ ID NO: 1 is substituted with Tyr (66Tyr), or        Asn (66Asn);    -   position 109 of SEQ ID NO: 1 is substituted with Glu (109Glu);    -   position 113 of SEQ ID NO: 1 is substituted with Thr (113Thr);    -   position 125 of SEQ ID NO: 1 is substituted with Phe (125Phe);    -   position 140 of SEQ ID NO: 1 is substituted with Val (140Val);    -   position 144 of SEQ ID NO: 1 is substituted with Arg (144Arg);    -   position 145 of SEQ ID NO: 1 is substituted with Gly (145Gly);    -   position 195 of SEQ ID NO: 1 is substituted with Gln (195Gln) or        Leu (195Leu);    -   position 197 of SEQ ID NO: 1 is substituted with Ile (197Ile);    -   position 217 of SEQ ID NO: 1 is substituted with Val (217Val);    -   position 223 of SEQ ID NO: 1 is substituted with Val (223Val);    -   position 232 of SEQ ID NO: 1 is substituted with Cys (232Cys),        Tyr (232Tyr), or Trp (232Trp), especially 232Cys;    -   position 239 of SEQ ID NO: 1 is substituted with Tyr (239Tyr),        or Trp (239Trp);    -   position 250 of SEQ ID NO: 1 is substituted with Met (250Met);        and/or    -   position 257 of SEQ ID NO: 1 is substituted with Met (257Met),        or Gln (257Gln).

Moreover, the above substitutions may be combined with one or more ofthe following substitutions:

-   -   position 37 of SEQ ID NO: 1 is substituted with Tyr (37Tyr);    -   position 202 of SEQ ID NO: 1 is substituted with Gly (202Gly),        and/or    -   position 230 of SEQ ID NO: 1 is substituted with Leu (230Leu),        wherein these are preferably combined with 250Met.

In a particularly preferred embodiment of the present invention,

-   -   a) the mutant 3-HBDH has at least the mutations 125Phe, 144Arg,        232Cys, and/or 250Met, particularly 125Phe, 144Arg, and/or        250Met, more particularly 250Met corresponding to positions of        SEQ ID NO:1; or    -   b) the mutant 3-HBDH has the mutations        -   125Phe;        -   144Arg;        -   232Cys;        -   250Met;        -   230Leu and 250Met;        -   37Tyr and 250Met;        -   37Tyr, 230Leu and 250Met;        -   202Gly, 230Leu and 250Met;        -   37Tyr, 145Gly, 230Leu and 250Met; and/or        -   37Tyr, 195Gln, 230Leu and 250Met        -   corresponding to positions of SEQ ID NO:1; or    -   c) the mutant 3-HBDH has only the mutations        -   125Phe;        -   144Arg;        -   232Cys;        -   250Met;        -   230Leu and 250Met;        -   37Tyr and 250Met;        -   37Tyr, 230Leu and 250Met;        -   202Gly, 230Leu and 250Met;        -   37Tyr, 145Gly, 230Leu and 250Met; or        -   37Tyr, 195Gln, 230Leu and 250Met        -   corresponding to positions of SEQ ID NO:1.

In a even more particularly preferred embodiment of the presentinvention, the mutant comprises an amino acid sequence that is at least80% identical to the amino acid sequence of SEQ ID NO: 1 (3-HBDH fromRhodobacter sphaeroides; wild-type 3-HBDH), has at least one amino acidsubstitution relative to the wild-type 3-HBDH at a positioncorresponding to position 250, preferably 250Met, of SEQ ID NO: 1 and

-   -   a) has at least the mutations 125Phe, 144Arg, and/or 232Cys,        particularly 125Phe and/or 144Arg, corresponding to positions of        SEQ ID NO:1; or    -   b) the mutant 3-HBDH has the mutations        -   125Phe;        -   144Arg;        -   232Cys;        -   250Met;        -   230Leu and 250Met;        -   37Tyr and 250Met;        -   37Tyr, 230Leu and 250Met;        -   202Gly, 230Leu and 250Met;        -   37Tyr, 145Gly, 230Leu and 250Met; and/or        -   37Tyr, 195Gln, 230Leu and 250Met        -   corresponding to positions of SEQ ID NO:1; or    -   c) the mutant 3-HBDH has only the mutations        -   250Met;        -   230Leu and 250Met;        -   37Tyr and 250Met;        -   37Tyr, 230Leu and 250Met;        -   202Gly, 230Leu and 250Met;        -   37Tyr, 145Gly, 230Leu and 250Met; or        -   37Tyr, 195Gln, 230Leu and 250Met        -   corresponding to positions of SEQ ID NO:1.

In a preferred embodiment of the present invention, the mutant 3-HBDHcomprises or consists of an amino acid sequence that is at least 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofany of SEQ ID NO: 1 to 11, particularly wherein the mutant 3-HBDHcomprises or consists of the sequence selected from the group consistingof SEQ ID NOs: 2 to 11.

As detailed above, the sequence of SEQ ID NO: 1 relates to the wild-type3-HBDH. The sequences of SEQ ID NO: 2 to 11 are the sequences ofpreferred mutants. The sequences of SEQ ID NO: 5 to 11 are the sequencesof highly preferred mutants. The sequences of the mutants and thesubstitutions effected are shown in the section “Sequences”. Sequenceidentity may be determined as described above.

In a preferred embodiment of the present invention, improved performancerelative to the wild-type 3-HBDH is

-   -   increased stability, especially thermal stability, relative to        the wild-type 3-HBDH; and/or    -   increased substrate affinity, especially for 3-hydroxybutyrate,        relative to the wild-type 3-HBDH; and/or    -   increased cofactor affinity, especially for NAD or a derivative        thereof, particularly wherein the derivate is carba-NAD,        relative to the wild-type 3-HBDH.

The term “increased stability, especially thermal stability, relative tothe wild-type 3-HBDH” means that the mutant 3-HBDH is less prone to lossof (enzyme) activity at a certain condition, especially at elevatedtemperatures. Stabilization of enzymes including avoidance ofdenaturation mechanisms in order to maintain their full potential ascatalysts is an important goal in biotechnology. Enzyme stabilizationhas notable importance due to increasing number of enzyme applications.The increase in stability allows for sustained usability (e.g. longerstorage, usability for a longer time etc). Increased stability of themutant relative to the wild-type can be determined by comparing theremaining activity of both enzymes (wild-type and mutant), e.g. afterstorage or exposure to a particular condition (e.g. elevatedtemperature, drying, buffer, or salt) (absolute remaining activity).Alternatively, the stability is improved compared to the wild-type, ifthe mutant, e.g., has a higher relative remaining activity in converting3-HB into acetoacetate. Relative remaining activity may be determined bycomparing the remaining or residual acidity after storage at givenconditions (e.g. storage time, temperature) to the initial activitybefore storage. Alternatively, stability is improved, if the mutant,e.g., has a higher remaining activity in converting 3-HB intoacetoacetate than the wild type enzyme. Relative remaining activity maybe determined by comparing the remaining or residual activities of wildtype and mutant enzyme after storage at given conditions (e.g. storagetime, temperature).

The term enzyme activity and its determination are well-known to theperson skilled in the art. Enzyme activity is generally defined asconversion of amount of substrate per time. The SI unit for enzymeactivity is katal (1 katal=1 mol s⁻¹). A more practical and commonlyused value is enzyme unit (U)=1 μmol min⁻¹. 1 U corresponds to 16.67nanokatals and is defined as the amount of the enzyme that catalyzes theconversion of 1 micro mole of substrate per minute. The specificactivity of an enzyme is the activity of an enzyme per milligram oftotal protein (expressed in μmol min⁻¹ mg⁻¹).

The enzyme activity may be determined in an assay measuring either theconsumption of substrate or cofactor or the formation of product overtime. A large number of different methods of measuring theconcentrations of substrates and products exist and many enzymes can beassayed in several different ways as known to the person skilled in theart. In the present invention, the 3-HBDH in question is, e.g.,incubated with 3-hydroxybutyrate and cofactor (e.g. NAD or derivativethereof such as carba-NAD) and the conversion of the cofactor (NAD toNADH or carba-NAD to carba-NADH) is monitored. Monitoring can, e.g., bedone by measuring light absorbance at 340 nm. The obtained change ofabsorption per minute (dE/min) represents the enzymatic activity. Fordetails see Example 2.

In a preferred embodiment of the present invention, increased stabilityof the mutant 3-HBDH relative to the respective 3-HBDH without mutationmay be expressed as increase in half-life of the mutant(t_(1/2)(mutant)) relative to the half-life of to the respectivewild-type 3HBDH (t_(1/2)(wild-type)). The half-life (t_(1/2)) of theenzyme indicates the amount of time in which 50% of the originalactivity (activity at t=0) is lost and after which the remainingactivity amounts to 50%. Accordingly, an increased stability results inan increased half-life of the mutant relative to the respectivewild-type. Increase in stability may be determined ast_(1/2)(mutant)/t_(1/2)(wild-type). Percental increase in half-life maybe determined as [t_(1/2)(mutant)/t_(1/2)(wild-type)−1]*100.

In a highly preferred embodiment of the present invention, increasedstability of the mutant 3-HBDH relative to the respective 3-HBDH withoutmutation may be determined and expressed as remaining activity after astress incubation (e.g. 30 min at e.g. 64° C. or any other conditiongiven in the Examples) in relation to the initial activity before stressincubation/storage (see Examples). For this, the enzymatic reaction maybe monitored (e.g. at room temperature at 340 nm for 5 minutes) and thechange in absorption per time (e.g. dE/min) may be calculated for eachsample. The values obtained for stressed samples may be compared to therespective un-stressed sample (value set to 100% activity) andcalculated in percent activity ((dE/min (stressed sample)/dE/min(unstressed sample)*100). Accordingly, a mutant's value higher than thevalue obtained with wild-type enzyme represents an improvement inthermal stability. The stability is increased, if [% remaining activityof the mutant]−[% remaining activity of the wild-type]>0. Alternatively,the remaining activity of the mutant may be also expressed as activityin percent and may be calculated as follows: [% remaining activity ofthe mutant]/[% remaining activity of the wild-type]*100%. The stabilityof the mutant relative to the wild-type is increased if the resultingvalue is >100%. A particular suitable test for determining stability isdescribed in detail in Example 2. In accordance with this, the stabilitymay be expressed as remaining activity and calculated as [dE/min(stressed sample)]/[dE/min (not stressed sample)]*100. Further detailsare given in Example 2. A value obtained with a mutant higher than thevalue obtained with wild-type enzyme represents an increased stabilityof the mutant.

Preferably, stability is increased by at least 10%, 20%, 30% or 40%,preferably at least 50%, 75% or 100%, still more preferably at least125%, 150%, 175% or 200%, especially 250% and most preferably 300%.

Moreover, there are commercial advantages in carrying out enzymaticreactions at higher temperatures. Accordingly, thermal stability of themutant is preferably increased. This means that resistance of the mutantrelative to the wild-type to increased temperatures is higher. Increasein thermal stability may be particularly determined as shown in theExamples, e.g. in Examples 2 and 3. In general, the mutant and thewild-type enzyme may be preincubated at an increased temperature (e.g.above 50° C. such as 54° C. or 64° C.) for a defined time (such as 10min or 30 min), after which the remaining activity in converting 3-HBinto acetoacetate of the mutant is compared to the remaining activity ofthe wild-type enzyme. If the remaining activity of the mutant is higherthan that of the wild-type, the mutant has an increased thermalstability.

A suitable method for the determination of increased thermal stabilityis detailed in the Examples. Exemplary conditions for stress conditionsmay be preincubation at 50-90° C., in particular at 50-64° C. (e.g., 50°C. or 54° C. or 64° C.) for 30 min and testing afterwards with 62.22 mM3-hydroxybutyrate; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes pH 9.0or 150 mM 3-hydroxybutyrate; 5 mM cNAD; 0.1% Triton X-100; 70 mM Mops pH7.5.

The term “increased substrate affinity, especially for3-hydroxybutyrate, relative to the wild-type 3-HBDH” means that theaffinity of the mutant for the substrate 3-hydroxybutyricacid/3-hydroxybutyrate/3-hydroxybutanoate (3-HB) which is converted intoacetoacetate is increased. For the determination, the enzymatic reactionmay be monitored (e.g. at room temperature at 340 nm for 5 minutes) andthe dE/min may be calculated for each sample. The affinity of the mutantis increased compared to the wild-type, if the mutant, e.g., has ahigher absolute or relative affinity for the substrate, particularly3-HB. Affinity of the mutant compared to the wild-type can be determinedby comparing the absolute affinities of both enzymes (wild-type andmutant) (absolute comparison) and may be calculated in percent activity((dE/min (mutant)/dE (wild-type))*100) (%). Alternatively, the affinityof the mutant compared to the wild-type can be determined by comparingthe relative affinities of both enzymes (wild-type and mutant) (relativecomparison). Relative affinity of wild-type or mutant may be determinedby setting the affinity at subsaturation substrate concentration inrelation to the affinity at saturation substrate concentration. Asdetailed in the Examples 2 and 3, affinity to 3-hydroxybutyrate may bedetermined in an activity assay with reduced amount of substrate (i.e.at subsaturation concentration), e.g. with 1.94 mM 3-hydroxybutyrate(further exemplary conditions: 4.15 mM cNAD; 0.1% Triton X-100; 200 mMHepes pH 9.0). A particular suitable test for determining affinity isdescribed in detail in Example 2. In accordance with this, the affinitymay be expressed as relative activity and calculated as [dE/min(subsaturation substrate concentration)]/[dE/min (saturation substrateconcentration)]*100. Further details are given in Example 2. A valueobtained with a mutant higher than the value obtained with wild-typeenzyme represents an increase in affinity for the mutant.

An increased affinity correlates with a lower Km value. The Michaelisconstant Km is the substrate concentration at which an enzyme reactionrate is at half-maximum and is an inverse measure of the substrate'saffinity for the enzyme.

The term “increased cofactor affinity, especially for NAD or aderivative thereof, particularly wherein the derivate is carba-NAD,relative to the wild-type 3-HBDH” means that the affinity of the mutantfor the cofactor (NAD or a derivate thereof, especially carba-NAD)needed to convert 3-HB into acetoacetate is increased. As detailed inthe Examples 2 and 3, affinity to the cofactor may be determined in anactivity assay with reduced amount of cofactor (i.e. below saturation),e.g. with 0.032 mM cNAD (further exemplary conditions: 62.22 mM3-hydroxybutyrate; 0.1% Triton X-100; 200 mM Hepes pH 9.0) or 0.5 mMcNAD. The above details given with respect to substrate affinity areanalogously applicable to the cofactor affinity. A particular suitabletest for determining affinity is described in detail in Example 2.

Particularly, the mutant 3-HBDH of the present invention ischaracterized in that the mutant 3-HBDH has an at least 2-fold increasedstability relative to the wild-type 3-HBDH, preferably an at least3-fold increased stability, preferably an at least 4-fold increasedstability, more preferably an at least 5-fold increased stability;and/or characterized in that the mutant 3-HBDH has an increasedsubstrate and/or cofactor affinity relative to the wild-type 3-HBDH,particularly an increased affinity for (i) 3-hydroxybutyrate and/or (ii)nicotinamide adenine dinucleotide (NAD) or a functionally activederivative thereof and/or particularly wherein the substrate and/orcofactor affinity is increased by at least 5%, more particularly atleast 10%, still more particularly by at least 15% or 20%.

In another aspect, the present invention relates to a nucleic acidencoding the mutant 3-HBDH of the present invention as described above.

The term “nucleic acid” as used herein generally relates to anynucleotide molecule which encodes the mutant 3-HBDH of the invention andwhich may be of variable length. Examples of a nucleic acid of theinvention include, but are not limited to, plasmids, vectors, or anykind of DNA and/or RNA fragment(s) which can be isolated by standardmolecular biology procedures, including, e.g. ion-exchangechromatography. A nucleic acid of the invention may be used fortransfection or transduction of a particular cell or organism.

Nucleic acid molecule of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA e.g. obtained by cloning or produced bychemical synthetic techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.Nucleic acid molecule as used herein also refers to, among other,single- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA.

Additionally, the nucleic acid may contain one or more modified bases.Such nucleic acids may also contain modifications e.g. in theribose-phosphate backbone to increase stability and half life of suchmolecules in physiological environments. Thus, DNAs or RNAs withbackbones modified for stability or for other reasons are “nucleic acidmolecule” as that feature is intended herein.

Moreover, DNAs or RNAs comprising unusual bases, such as inosine, ormodified bases, such as tritylated bases, to name just two examples, arenucleic acid molecule within the context of the present invention. Itwill be appreciated that a great variety of modifications have been madeto DNA and RNA that serve many useful purposes known to those of skillin the art. The term nucleic acid molecule as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof nucleic acid molecule, as well as the chemical forms of DNA and

RNA characteristic of viruses and cells, including simple and complexcells, inter alia.

Furthermore, the nucleic acid molecule encoding the mutant 3-HBDH of theinvention can be functionally linked, using standard techniques such asstandard cloning techniques, to any desired sequence, such as aregulatory sequence, leader sequence, heterologous marker sequence or aheterologous coding sequence to create a fusion protein.

The nucleic acid of the invention may be originally formed in vitro orin a cell in culture, in general, by the manipulation of nucleic acidsby endonucleases and/or exonucleases and/or polymerases and/or ligasesand/or recombinases or other methods known to the skilled practitionerto produce the nucleic acids.

The nucleic acid of the invention may be comprised in an expressionvector, wherein the nucleic acid is operably linked to a promotersequence capable of promoting the expression of the nucleic acid in ahost cell.

As used herein, the term “expression vector” generally refers to anykind of nucleic acid molecule that can be used to express a protein ofinterest in a cell (see also above details on the nucleic acids of thepresent invention). In particular, the expression vector of theinvention can be any plasmid or vector known to the person skilled inthe art which is suitable for expressing a protein in a particular hostcell including, but not limited to, mammalian cells, bacterial cell, andyeast cells. An expression construct of the present invention may alsobe a nucleic acid which encodes a 3-HBDH of the invention, and which isused for subsequent cloning into a respective vector to ensureexpression. Plasmids and vectors for protein expression are well knownin the art, and can be commercially purchased from diverse suppliersincluding, e.g., Promega (Madison, Wis., USA), Qiagen (Hilden, Germany),Invitrogen (Carlsbad, Calif., USA), or MoBiTec (Germany). Methods ofprotein expression are well known to the person skilled in the art andare, e.g., described in Sambrook et al., 2000 (Molecular Cloning: Alaboratory manual, Third Edition).

The vector may additionally include nucleic acid sequences that permitit to replicate in the host cell, such as an origin of replication, oneor more therapeutic genes and/or selectable marker genes and othergenetic elements known in the art such as regulatory elements directingtranscription, translation and/or secretion of the encoded protein. Thevector may be used to transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. The vector optionally includes materials toaid in achieving entry of the nucleic acid into the cell, such as aviral particle, liposome, protein coating or the like. Numerous types ofappropriate expression vectors are known in the art for proteinexpression, by standard molecular biology techniques. Such vectors areselected from among conventional vector types including insects, e.g.,baculovirus expression, or yeast, fungal, bacterial or viral expressionsystems. Other appropriate expression vectors, of which numerous typesare known in the art, can also be used for this purpose. Methods forobtaining such expression vectors are well-known (see, e.g. Sambrook etal, supra).

As detailed above, the nucleic acid which encodes a mutant 3-HBDH of theinvention is operably linked to sequence which is suitable for drivingthe expression of a protein in a host cell, in order to ensureexpression of the protein. However, it is encompassed within the presentinvention that the claimed expression construct may represent anintermediate product, which is subsequently cloned into a suitableexpression vector to ensure expression of the protein. The expressionvector of the present invention may further comprise all kind of nucleicacid sequences, including, but not limited to, polyadenylation signals,splice donor and splice acceptor signals, intervening sequences,transcriptional enhancer sequences, translational enhancer sequences,drug resistance gene(s) or alike. Optionally, the drug resistance genemay be operably linked to an internal ribosome entry site (IRES), whichmight be either cell cycle-specific or cell cycle-independent.

The term “operably linked” as used herein generally means that the geneelements are arranged as such that they function in concert for theirintended purposes, e.g. in that transcription is initiated by thepromoter and proceeds through the DNA sequence encoding the protein ofthe present invention. That is, RNA polymerase transcribes the sequenceencoding the fusion protein into mRNA, which in then spliced andtranslated into a protein.

The term “promoter sequence” as used in the context of the presentinvention generally refers to any kind of regulatory DNA sequenceoperably linked to a downstream coding sequence, wherein said promoteris capable of binding RNA polymerase and initiating transcription of theencoded open reading frame in a cell, thereby driving the expression ofsaid downstream coding sequence. The promoter sequence of the presentinvention can be any kind of promoter sequence known to the personskilled in the art, including, but not limited to, constitutivepromoters, inducible promoters, cell cycle-specific promoters, and celltype-specific promoters.

Another aspect of the present invention relates to a cell comprising themutant 3-HBDH of the present invention or the nucleic acid of thepresent invention. In one embodiment of the present invention, a cellcomprising the mutant is used in the context of the present invention.The cell is preferably a host cell. A “host cell” of the presentinvention can be any kind of organism suitable for application inrecombinant DNA technology, and includes, but is not limited to, allsorts of bacterial and yeast strain which are suitable for expressingone or more recombinant protein(s). Examples of host cells include, forexample, various Bacillus subtilis or E. coli strains. A variety of E.coli bacterial host cells are known to a person skilled in the art andinclude, but are not limited to, strains such as DH5-alpha, HB101,MV1190, JM109, JM101, or XL-1 blue which can be commercially purchasedfrom diverse suppliers including, e.g., Stratagene (CA, USA), Promega(WI, USA) or Qiagen (Hilden, Germany). A particularly suitable host cellis also described in the Examples, namely E. coli XL-1 Blue cells.Bacillus subtilis strains which can be used as a host cell include,e.g., 1012 wild type: leuA8 metB5 trpC2 hsdRM1 and 168 Marburg: trpC2(Trp−), which are, e.g., commercially available from MoBiTec (Germany).

The cultivation of host cells according to the invention is a routineprocedure known to the skilled person. That is, a nucleic acid encodinga mutant 3-HBDH of the invention can be introduced into a suitable hostcell(s) to produce the respective protein by recombinant means. Thesehost cells can by any kind of suitable cells, preferably bacterial cellssuch as E. coli, which can be easily cultivated. At a first step, thisapproach may include the cloning of the respective gene into a suitableplasmid vector. Plasmid vectors are widely used for gene cloning, andcan be easily introduced, i.e. transformed, into bacterial cells whichhave been made competent. After the protein has been expressed in therespective host cell, the cells can be broken by means of eitherchemical or mechanical cell lysis are well known to the person skilledin the art, and include, but are not limited to, e.g. hypotonic salttreatment, detergent treatment, homogenization, or ultrasonification.

In another aspect the present invention relates to a method ofdetermining the amount or concentration of 3-hydroxybutyrate in asample, the method comprising

-   a) contacting the sample with the mutant 3-HBDH of the present    invention under conditions conducive to the activity of the 3-HBDH;-   b) reacting 3-hydroxybutyrate with nicotinamide adenine dinucleotide    (NAD) or a functionally active derivative thereof; and-   c) determining the change in the redox state of NAD or the    derivative thereof, thereby determining the amount or concentration    of 3-hydroxybutyrate in the sample.

The above method is based on the fact that 3-HBDH may be used tocatalyze the conversion of 3-HB to acetoacetate according to thefollowing scheme:(R)-3-hydroxybutyrate+NAD⁺

acetoacetate+NADH+H⁺

In a first step of the method of the present invention a sample iscontacted with the 3-HBDH of the present invention. The contacting ofthe sample with the mutant 3-HBDH can be direct (e.g. in liquid assays)or indirect (e.g. in sensor systems in which only a fraction of thesample (containing the analyte) is contacting the 3-HBDH).

It is evident that the contacting should be carried out under conditionsconducive to the activity of the 3-HBDH, i.e. allowing the enzyme toconvert 3-HB to acetoacetate. Incubation step can vary from about 5seconds to several hours, preferably from about 10 seconds to about 10minutes. However, the incubation time will depend upon the assay format,volume of solution, concentrations and the like. Usually the assay willbe carried out at ambient temperature or a temperature required forother test formats carried out concomitantly (e.g. 25° C. to 38° C.;such as 30° C. or 37° C.), although it can be conducted over a range oftemperatures, such as 10° C. to 40° C.

Optionally, the enzyme can be fixed to or immobilized into a supportlayer prior to the contacting with the sample to facilitate the assay.Examples of support layers include glass or plastic in the form of, forexample, a microtiter plate, a glass microscope slide or cover slip, astick, a bead, or a microbead, membranes (e.g. used in test strips) andlayers of biosensors.

The sample may be any sample suspected of containing 3-HB, particularlya sample from a subject. The term “sample from a subject” includes allbiological fluids, excretions and tissues isolated from any givensubject, particularly a human. In the context of the present inventionsuch samples include, but are not limited to, blood, blood serum, bloodplasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma,prostatic fluid, excreta, tears, saliva, sweat, biopsy, ascites,cerebrospinal fluid, milk, lymph, bronchial and other lavage samples, ortissue extract samples. Preferably, the subject is an animal (includinghuman), more preferably a mammal, still more preferably a human.Preferably, the sample is a body fluid, particularly a blood sample or aurine sample.

Typically, blood samples are preferred test samples for use in thecontext of the present invention. For this, blood may be drawn from avein, usually from the inside of the elbow or the back of the hand or afingertip. Particularly, in infants or young children, a sharp toolcalled a lancet may be used to puncture the skin and make it bleed. Theblood may be collected e.g. into a pipette or cannula, or onto a slideor test strip.

After the contacting and the conversion of 3-HB, if present, the changein the redox state of NAD or derivate mediated by the 3-HBDH aredetermined, thereby determining 3-HB in the sample. Evidently, theamount of NADH or derivate thereof produced and the amount of NAD orderivate thereof consumed correlate with the amount of 3-HB present inthe sample. Accordingly, the change in the redox state of NAD includesthe determination of the amount or concentration of NAD and/or NADH aswell as the ratio of the two. The same applies to NAD derivates.

A variety of methods for determining NADH/NAD or derivate thereof areknown in the art and any of these can be used.

Exemplary methods for determining NADH/NAD or derivate thereof includeelectrochemical methods (e.g. as described in U.S. Pat. No. 6,541,216)or optical methods (e.g. by measuring NAD/NADH conversion by lightabsorbance at e.g. 340 nm or 365 nm or by assays based on a reductase toform luciferin, which is then quantified optically). If electrochemicalmethods are used, NADH/NAD or derivate thereof can either a) reactdirectly on a measurement electrode or b) NADH/NAD or derivate thereofreacts in a first step with an additional redoxmediator substance whichchanges its redox state in a defined relation to the redox state ofNADH/NAD or derivate thereof and this redoxmediator reacts in asubsequent step on the measurement electrode.

Both NAD and NADP are base-labile molecules the degradation paths ofwhich are described in the literature (see e.g. N.J.Oppenheimer in ThePyridine Nucleotide Coenzymes Academic Press, New York, London 1982, J.Everese, B. Anderson, K. Yon, Editors, chapter 3, pages 56-65).Therefore, derivatives of NADH/NAD have been developed and arecommercially available. Some derivates are described in the PyridineNucleotide Coenzymes, Academic Press New York, London 1982, Eds. J.Everese, B. Anderson, K. You, Chapter 4, WO 01/94370, WO 98/33936 andU.S. Pat. No. 5,801,006.

Preferably, carba-NAD (cNAD) is used as a derivative of NAD. Incarba-NAD the ribose is substituted by a carbacyclic sugar unit.Carba-NAD has the following structure (I):

The compound, its production and use are described in detail in WO2007/012494, WO 2011/012270 and WO2014/195363. The cofactor in thepresent invention is preferably carba-NAD. In one embodiment of thepresent invention, the cofactor is a functionally active derivative ofNAD as disclosed in formula III of WO 2011/012270 to which it isexplicitly referred. In one embodiment of the present invention, NADP isused instead of NAD.

The method of the present invention can be carried out in a so-calledliquid or wet test, for example in a cuvette, or as a so-called dry teston an appropriate reagent carrier, the necessary test reagents therebybeing present in or on a solid carrier, which is preferably an absorbentor swellable material.

Alternatively or additionally, the 3-HBDH may be part of a sensor, atest strip, a test element, a test strip device or a liquid test.

A sensor is an entity that measures a physical/chemical quantity andconverts it into a signal which can be read by an observer or by aninstrument. In the present invention, the 3-HBDH may be part of asensor. The sensor converts 3-HB and NAD or a derivate thereof intoacetoacetate and NADH or a derivate thereof, which is further convertedinto a signal such as a change in colour or a value displayed e.g. on adisplay or monitor.

In one embodiment, the sensor may comprise 3-HBDH and an amperometricdevice to determine 3-HB of a sample. Also, a microdialysis systemcoupled with an electrochemical flow cell could be used for continuousmonitoring of 3-HB in a sample or subject. The working electrode of theflow cell could be prepared with the 3-HBDH immobilized in a redoxpolymer film on the electrode surface. Coupling an electrochemical 3-HBsensor directly with microdialysis eliminates the need to transfersample aliquots to a liquid chromatography system with a post-columnoxidase enzyme reactor. 3-HB in the dialysate from the microdialysisprobe can be selectively detected at the enzyme electrode without anysignificant interference from other oxidizable species. Furthermore,enzyme-coupled biosensors have been described in the art. In accordancewith this, 3-HBDH may be coupled to a surface (e.g. by printing a3-HBDH/graphite mixture onto electroplated graphite pads or byadsorption or immobilization of the mutant 3-HBDH on carbon particles,platinized carbon particles, carbon/manganese dioxide particles, glassycarbon, or mixing it with carbon paste electrodes etc.)

A test strip or a test element is an analytic or diagnostic device usedto determine presence and/or quantity of a target substance within asample. A standard test strip may comprise one or more differentreaction zones or pads comprising reagents which react (e.g. changecolour) when contacted with a sample. Test strips are known in manyembodiments, for example from U.S. Pat. No. 6,541,216, EP 262445 andU.S. Pat. No. 4,816,224. It is commonly known that one or more reagents(e.g. enzymes) needed for carrying out the determination methods arepresent on or in solid carrier layers. As carrier layers, there areespecially preferred absorbent and/or swellable materials which arewetted by the sample liquid to be analyzed. Examples include gelatine,cellulose and synthetic fiber fleece layers.

The 3-HBDH of the present invention may also be part of a liquid test. Aliquid test is a test wherein test components react in a liquid medium.Usually in the field of laboratory analytics, the liquid reagents are onwater basis, e.g. a buffered salt solution in order to provide theactivity of enzyme(s) involved. The liquid is usually adapted to thespecific intended use. For carrying out a liquid test, all testcomponents are solved in a liquid and combined (or vice versa). Typicalcontainments for carrying out such tests include vials, multi wellsplates, cuvettes, vessels, reagent cups, tubes etc.

In one embodiment of the present invention, the 3-HBDH of the presentinvention may be immobilized. Typical methods of immobilization includecovalent binding e.g. to a membrane, encapsulation in a polymer,cross-linking to a supporting matrix or immobilization in a sol-gelmatrix (e.g. glasses such as silicate glasses) or adsorption on poroussubstrates. Suitable methods for immobilizing enzymes are known in theart (see e.g. Lillis et al., 2000, Sensors and Actuators B 68: 109-114).

In a preferred embodiment of the present invention, the method furthercomprises determining the amount or concentration of glucose. In anotherembodiment of the present invention the method comprises determining theamount or concentration of acetone and/or acetoacetate; and/ordetermining the amount or concentration of glucose. The determination ofthese compounds is of particular relevance in the diagnosis of the abovediseases and medical condition. Methods for determining these compoundsare well-known in the art. Additionally, systems and methods formultiple analyte analysis are known from WO 2014/068024.

Accordingly and preferably, the method of the present invention isfurther characterized in that

-   a) wherein the determining of the change in the redox state of NAD    or the derivate thereof includes the determination of the    concentration of (i) NAD or the derivate thereof and/or (ii) NADH or    the derivate thereof; and/or-   b) wherein the determining the change in the redox state of NAD or    the derivative thereof is electrochemically or optically; and/or-   c) wherein the method further comprises determining the amount or    concentration of acetoacetate and/or acetone; and/or-   d) wherein the method further comprises determining the amount or    concentration of glucose; and/or-   e) wherein the derivative of NAD is carba-NAD; and/or-   f) wherein the mutant 3-HBDH is part of a sensor, a test strip, a    test element, a test strip device or a liquid test; and/or-   g) wherein the sample is a body fluid, particularly a blood sample    or a urine sample.

In still another aspect, the present invention relates to the use of themutant 3-HBDH of the present invention for determining the amount orconcentration of 3-hydroxybutyrate in a sample.

With respect to the use of the present invention it is referred to theterms, examples and specific embodiments used in the context of theother aspects of the present disclosure, which are also applicable tothis aspect. Particularly, the mutant 3-HBDH according to the presentinvention may be used as detailed with respect to the methods of thepresent invention.

Yet, in another aspect, the present invention relates to a device fordetermining the amount or concentration of 3-hydroxybutyrate in a samplecomprising the mutant 3-HBDH of the present invention and optionally afurther component required for said determining.

With respect to the device of the present invention it is referred tothe terms, examples and specific embodiments used in the context of theother aspects of the present disclosure, which are also applicable tothis aspect. Particularly, the mutant 3-HBDH according to the presentinvention may be employed as detailed above.

The 3-HBDH of the present invention may be part of a device fordetermining 3-HB in a sample. The device may be any device suitable forthis purpose. The device may be a machine or tool which can be used fordetermining 3-HB. Preferably, the device is a sensor, preferably anelectrochemical sensor, or a test strip. Exemplary devices are describedabove and in the following:

The device may be a sensor, e.g. a biosensor, which is an analyticaldevice for the detection of an analyte that combines a biologicalcomponent (here the 3-HBDH according to the present invention) with adetector component, particularly a physicochemical detector component.

Biosensors are particularly useful to determine the concentration ofvarious analytes (including 3-HB) from biological samples, particularlyfrom blood. Exemplary biosensors based on an electrochemical test stripformat are described in U.S. Pat. Nos. 5,413,690; 5,762,770 and5,997,817.

In the (bio)sensor of the present invention, 3-HB converted intoacetoacetate in the presence of the 3-HBDH and NAD or derivative and thechange in the redox state of NAD or derivative may be monitored by thetransducer or detector element.

Particularly, 3-HB sensors have been combined with other sensors, e.g.for determining glucose, acetone, acetoacetate, cholesterol,triglycerides, urea, blood gases or electrolytes etc. Evidently, themutant 3-HBDH of the present invention could also be used in thesemulti-analyte devices.

As detailed above, the sensor is preferably an electrochemical oroptical sensor. An electrochemical sensor is based on the translation ofa chemical signal (here presence of 3-HB) into an electrical signal(e.g. current). A suitable electrode can measure the 3-HB-mediatedproduction of NADH or derivative thereof as an electrical signal.

A suitable optical sensor can measure the 3-HBDH-mediated change in theredox state of NAD or derivate thereof. The signal may be theNAD/NADH-mediated absorbance/emission of light.

The device of the present invention may comprise—in addition to the3-HBDH of the present invention—one or more further component(s), suchas other reagents, required for or helpful in said determining. Thecomponents may be any of these described in the context of the methodsand devices of the present invention. Additionally, this may include aninstruction manual, a lancet device, a capillary pipette, a furtherenzyme, a substrate and/or a control solution etc.

Preferably, the device of the present invention is characterized in thatthe device is or comprises a sensor, preferably an electrochemicalsensor or an optical sensor, or a test strip, particularly a test stripand/or allows for determining the amount or concentration of glucose inthe sample. With respect to the device of the present invention it isalso referred to the terms, examples and specific embodiments describedabove.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention.Definitions of common terms in molecular biology can be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The invention is not limited to the particular methodology, protocols,and reagents described herein because they may vary. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice of the present invention, the preferredmethods, and materials are described herein. Further, the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to limit the scope of the present invention.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Similarly, the words “comprise”, “contain” and “encompass”are to be interpreted inclusively rather than exclusively. Similarly,the word “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “plurality” refers to two or more.

The following Examples are intended to illustrate various embodiments ofthe invention. As such, the specific modifications discussed are not tobe construed as limitations on the scope of the invention. It will beapparent to the person skilled in the art that various equivalents,changes, and modifications may be made without departing from the scopeof the invention, and it is thus to be understood that such equivalentembodiments are to be included herein.

EXAMPLES Example 1 Establishing a Library of 3-HBDH Mutants

The gene for 3-hydroxybutyrate dehydrogenase (3-HBDH) from Rhodobactersphaeroides (Database UniProtKB-D0VWQ0) was synthesized, cloned invector pKKt5 and transformed in E. coli strain XL-1 Blue by commonmethods of molecular biology.

Saturation mutagenesis was applied on many amino acid positions of theenzyme. Mutagenesis was achieved by applying randomly synthesizedprimers with QuikChange Site-Directed Mutagenesis Kit (AgilentTechnologies Cat. 200518).

The 5′- and the 3′-primer used for mutagenesis were complementary toeach other and contained NNN (randomly synthesized nucleotides) for theamino acid exchange in a central position. This randomly created codonwas flanked by 12 to 16 nucleotides at each end. The sequences of thesenucleotides were identical to the cDNA-strand or to the complementarycDNA-strand flanking the codon for the amino substitution. Mutantlibrary was created by transformation of mutated genes in E. coli strainXI-Blue and cultivation on agar plates over night at 37° C.

Example 2 Determination of Properties of 3-HBDH Mutants from First Roundof Mutagenesis (Mutants with Single Amino Acid Substitutions)

A library of 3-HBDH mutants produced as described in Example 1 wasscreened for the following enzymatic properties:

-   -   Thermal stability    -   Affinity for 3-hydroxybutyrate    -   Affinity for c-NAD (carba-NAD=artificial cofactor cf.        US20120130062A1)

Mutant colonies on agar plates described above were picked in microtiterplates (mtp) containing 200 μl LB-Ampicillin-media/hole and incubated at37° C. over night. These plates were referred to as master plates. Foreach amino acid position two master plates were picked to assure thatevery possible exchange is included.

From each master plate, 40 μl sample/cavity was transferred to a mtpcontaining 200 μl 0.1% Triton X-100; 500 mM NaCl; 200 mM Hepes pH 9.0;2% B-Per/cavity (B-PER=Bacterial Protein Extraction Reagent Pierce No.78248) and incubated for cell disruption at 40° C. for 30 minutes. Thisplate was referred to as working plate.

From the working plate 4×20 μl sample/cavity was transferred to fourempty mtps. One of these was tested with 62.22 mM 3-hydroxybutyrate;4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes pH 9.0 at room temperatureand referred to as reference measurement. The other mtps were testedunder different conditions and the obtained values compared to thereference plate in percent.

Following parameters were measured:

-   -   Thermal stability: Mtp was incubated for 30 min. at 50° C.        (unless indicated otherwise) and tested afterwards with 62.22 mM        3-hydroxybutyrate; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes        pH 9.0    -   Affinity to 3-hydroxybutyrate: Activity assay with reduced        amount of substrate (i.e. below substrate saturation).        Measurement with 1.94 mM 3-hydroxybutyrate; 4.15 mM cNAD; 0.1%        Triton X-100; 200 mM Hepes pH 9.0    -   Affinity to cNAD: Activity assay with reduced amount of cofactor        (i.e. below saturation). Measurement with 62.22 mM        3-hydroxybutyrate; 0.032 mM cNAD; 0.1% Triton X-100; 200 mM        Hepes pH 9.0

The enzymatic reaction was monitored at room temperature at 340 nm for 5minutes and the dE/min calculated for each cavity in each working plate.The value from the reference measurement was set to 100% activity. Thevalues obtained with the other three plates (thermal stability, affinityto 3-hydroxybutyrate or cNAD) were compared to the reference andcalculated in percent activity ((dE/min Parameter/dE/minReference)*100). Each master plate contained beside the mutantswild-type enzyme as control to better estimate improvements ordeteriorations of the properties.

Thermal stability expressed as remaining activity was calculated asfollows:

${\left( \frac{\begin{matrix}{{dE}\text{/}\min\mspace{14mu}{stressed}\mspace{14mu}{sample}} \\\left( {{i.e.\mspace{14mu}{in}}\mspace{14mu}{example}\mspace{14mu} 2\text{:}\mspace{14mu} 30\mspace{14mu}{\min.\mspace{14mu} 50}{^\circ}\mspace{14mu}{C.}} \right)\end{matrix}}{{dE}\text{/}\min\mspace{14mu}{not}\mspace{14mu}{stressed}\mspace{14mu}{sample}} \right)*100} = {{remaining}\mspace{14mu}{activity}\mspace{14mu}{in}\mspace{14mu}{percent}}$

A value obtained with a mutant higher than the value obtained withwild-type enzyme represents an increase in thermal stability for themutant.

Substrate affinity expressed as activity ratio was calculated asfollows:

${\left( \frac{{dE}\text{/}\min\mspace{14mu}{obtained}\mspace{14mu}{with}\mspace{14mu}{less}\mspace{14mu}{substrate}}{{dE}\text{/}\min\mspace{14mu}{obtained}\mspace{14mu}{with}\mspace{14mu}{substrate}\mspace{14mu}{in}\mspace{14mu}{saturation}} \right)*100} = {{activity}\mspace{14mu}{in}\mspace{14mu}{percent}}$

A mutant with higher substrate affinity will show higher activity whenreacted with less substrate (below substrate saturation) than a mutantwith lower substrate affinity. A value obtained with a mutant higherthan the value obtained with wild-type enzyme represents an increase insubstrate affinity for the mutant.

Cofactor affinity expressed as activity ratio was calculatedaccordingly:

${\left( \frac{{dE}\text{/}\min\mspace{14mu}{obtained}\mspace{14mu}{with}\mspace{14mu}{less}\mspace{14mu}{cofactor}}{{dE}\text{/}\min\mspace{14mu}{obtained}\mspace{14mu}{with}\mspace{14mu}{cofactor}\mspace{14mu}{in}\mspace{14mu}{saturation}} \right)*100} = {{activity}\mspace{14mu}{in}\mspace{14mu}{percent}}$

Data below 0.001 dE/min were set to zero, resulting in “zero” values.

The results relative to wild type enzyme are summarized in Tables 1A and1B.

In a first round of saturation mutagenesis the following mutants werefound:

TABLE 1A Thermal stability and affinity of various single mutantsrelative to the wild-type 3-HBDH referred to as WT Affinity for ThermalClone 3-HB cNAD Stability WT + D2G + ∘ ∘ WT + D2S + ∘ + WT + N4T + nt ∘WT + N4C + nt ∘ WT + T11P + + + WT + N14T + nt ∘ WT + E23N + ∘ − WT +F37Y + nt + WT + E42C ∘ ∘ + WT + S57N + nt ∘ WT + S57C + nt + WT +S57Y + nt ∘ WT + S57P + nt + WT + A62P + ∘ ∘ WT + A62R ++ ∘ + WT + S66N∘ ∘ ++ WT + S66Y ∘ ∘ ++ WT + D67C ∘ ∘ + WT + E69M + ∘ + WT + E76D ∘ ∘ +WT + D87A − nt + WT + Q90T − nt + WT + H92Q ++ ∘ −− WT + S94A + nt +WT + S95R ++ ++ + WT + E97I + + − WT + E98T ∘ ∘ + WT + A106E ∘ ∘ + WT +A109E ∘ ∘ ++ WT + A109T ∘ ∘ ++ WT + N111I − nt + WT + S113T ∘ ∘ ++ WT +A115Y − − + WT + G125F + ∘ ++ WT + A128K ∘ ∘ + WT + A140V − − ++ WT +T144R + ∘ + WT + T144V − ∘ + WT + A145G ∘ ∘ + WT + P147R + + − WT +P147Q + + − WT + V165T − − + WT + V185I + + − WT + L186K + ∘ − WT +P188E − − + WT + P195Q − − ++ WT + P195L − − ++ WT + D196L − − + WT +Q197I − − ++ WT + A200K − nt + WT + A200L + nt ∘ WT + D202G + + + WT +D202N + + + WT + M203V + + − WT + T207R + + − WT + V208Y − nt ++ WT +V212I ++ ∘ − WT + Q217V − − ++ WT + A223V − − ++ WT + T225P ∘ ∘ + WT +G226K ∘ ∘ + WT + G230C ++ nt − WT + G230L ++ nt ∘ WT + V232C + ∘ ++ WT +V232Y ∘ ∘ ++ WT + V232W ∘ ∘ ++ WT + V232P + + − WT + A239V + ∘ − WT +A239Y ∘ ∘ ++ WT + A239W ∘ ∘ ++ WT + A239P + ∘ − WT + V250I ++ nt + WT +V250M + ∘ ++ WT + L257M ∘ ∘ ++ WT + L257Q − − ++ WT + L257M ∘ ∘ ++ (+ =improved; ∘ = similar; − = decreased; nt = not tested)

TABLE 1B Thermal stability and affinity of various single mutants of3-HBDH Affinity for* Thermal Clone 3-HB cNAD Stability** WT 33 9 16 WT +S66Y 34 10 100 WT + S66N 35 8 95 WT + A109E 26 5 99 WT + S113T 34 11 100WT + G125F 39 8 92 WT + A140V 13 4 100 WT + T144R 40 13 100 WT + A145G30 7 100 WT + P195Q 11 4 100 WT + P195L 10 5 96 WT + Q197I 21 5 100 WT +Q217V 22 3 100 WT + A223V 18 6 100 WT + V232C 41 11 100 WT + V232Y 31 4100 WT + V232W 30 4 100 WT + A239Y 31 4 100 WT + A239W 30 4 100 WT +V250M 40 10 100 WT + L257M 34 10 100 WT + L257Q 22 7 100 *given asactivity ratio (%) with 1.94/62.22 mM 3-HB and 0.032/4.15 mM cNAD**given as remaining activity (%) after a 30 minute-incubation at 50° C.

Table shows that often improvement of thermal stability has an effect onaffinity for substrate and/or cofactor. Four positions were found toimprove both stability and affinity parameters: Positions 125, 144, 232and 250 of SEQ ID NO: 1.

Exemplary mutant with exchange V250M was chosen for further optimizationby combining of additional found positions.

Example 3 Screening of 3-HBDH Mutants from Second Round of Mutagenesis(Mutants with Amino Acid Substitution V250M/I and Optionally FurtherAmino Acid Substitution(s))

Unless indicated otherwise, the experiments have been carried out asdetailed in Example 2. In a second round of mutagenesis selectedsubstitutions were combined into variant WT+V250M. The results aresummarized in Tables 2A and 2B:

TABLE 2A Thermal stability (54° C., 30 min) and affinity of variousmutants with amino acid substitution V250M or V250I Affinity for*Thermal Clone 3-HB cNAD Stability** WT 33 9 0 (5 50° C.) WT + V250I 41nt  0 (27 50° C.) WT + V250M 41 16 31 (83 50° C.) WT + G230L + V250M 378 75 WT + F37Y + V250M 40 17 33 WT + A140V + V250M 9 5 44 WT + F37Y +G230L + V250M 37 10 89 WT + F37Y + A140V + V250M 13 4 75 WT + A140V +G230L + V250M 13 5 80 WT + D202G + G230L + V250M 41 9 74 *given asactivity ratio (%) with 1.94/62.22 mM 3-HB and 0.032/4.15 mM cNAD**given as remaining activity (%) after a 30-minute incubation at 54° C.(unless indicated otherwise) nt not tested

Table 2A shows that multiple substitutions (in addition to V250M) show afurther increased stability. Mutants WT+G230L+V250M, WT+F37Y+V250M,WT+F37Y+G230L+V250M and WT+D202G+G230L+V250M are of particular interestas they also show good affinity for 3-HB.

Exemplary variant WT+F37Y+G230L+V250M was further combined with otherfound positions. The results are summarized in Table 2B:

TABLE 2B Thermal stability (64° C., 30 min) and affinity of variousmutants with amino acid substitutions F37Y, G230L and V250M Affinityfor* Thermal Clone 3-HB cNAD Stability** WT + F37Y + G230L + V250M 36 47 WT + F37Y + P195Q + G230L + V250M 13 4 51 WT + F37Y + A145G + G230L +V250M 34 11 29 *given as activity ratio (%) with 1.94/62.22 mM 3-HB and0.032/4.15 mM cNAD **given as remaining activity (%) after a 30-minuteincubation at 64° C.

Table 2B shows that stability of mutant WT+F37Y+G230L+V250M can befurther increased by adding substitution P195Q or A145G, which show alsoacceptable affinity for 3-HB and cNAD.

SEQUENCESWT: wild-type 3-HBDH from Rhodobacter sphaeroides (SEQ ID NO: 1)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTISV DGGWTAL 257 WT + G125F (SEQ ID NO: 2)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALP F MRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTISV DGGWTAL 257 WT + T144R (SEQ ID NO: 3)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGL R ASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTISV DGGWTAL 257 WT + V232C (SEQ ID NO: 4)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG T C VFLCSGAA 240DQITGTTISV DGGWTAL 257 WT + V250M (SEQ ID NO: 5)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTIS M  DGGWTAL 257 WT + G230L + V250M (SEQ ID NO: 6)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIG L  TVVFLCSGAA 240DQITGTTIS M  DGGWTAL 257 WT + F37Y + V250M (SEQ ID NO: 7)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINS Y TDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIGG TVVFLCSGAA 240DQITGTTIS M  DGGWTAL 257 WT + F37Y + G230L + V250M (SEQ ID NO: 8)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINS Y TDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIG L  TVVFLCSGAA 240DQITGTTIS M  DGGWTAL 257 WT + D202G + G230L + V250M (SEQ ID NO: 9)MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINSFTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA H G MDRETVIR EVMLDRQPSR QFATTGQIG L  TVVFLCSGAA240 DQITGTTIS M  DGGWTAL 257 WT + F37Y + A145G + G230L +V250M (SEQ ID NO: 10) MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINS YTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLT G SPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQIPDQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIG L  TVVFLCSGAA 240DQITGTTIS M  DGGWTAL 257 WT + F37Y + P195Q + G230L +V250M (SEQ ID NO: 11) MDLNGKRAIV TGSNSGIGLG CAEELARAGA EVVINS YTDR DEDHALAEKI GREHGVSCRY  60IAADMSDGEA CRALIETAGG CDILVNNAGI QHVSSIEEFP VGKWNAILAI NLSSAFHTTA 120AALPGMRAKG WGRIVNIASA HGLTASPYKS AYVAAKHGVV GFTKVTALET AGKGITCNAI 180CPGYVLTPLV EAQI Q DQMKA HDMDRETVIR EVMLDRQPSR QFATTGQIG L  TVVFLCSGAA240 DQITGTTIS M  DGGWTAL 257

The invention claimed is:
 1. A mutant 3-hydroxybutyrate dehydrogenase(3-HBDH) from Rhodobacter sphaeroides with improved performance relativeto the wild-type 3-HBDH, wherein the mutant comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 1 (wild type 3-HBDH from Rhodobacter sphaeroides) and whereinthe mutant has at least one amino acid substitution relative to thewild-type 3-HBDH at a position corresponding to position 250 of SEQ IDNO: 1, wherein, the improved performance is at least increased thermalstability relative to the wild-type 3-HBDH.
 2. The mutant 3-HBDH ofclaim 1, wherein the amino acid at the position corresponding toposition 250 of SEQ ID NO: 1 is substituted with Met (250Met) or Ile(250Ile).
 3. The mutant 3-HBDH of claim 1, wherein the mutant has atleast one further amino acid substitution at one or more of theposition(s) corresponding to position(s) 66, 109 113, 125, 140, 144,145, 195, 197, 217, 223, 232, 239 and/or 257 of SEQ ID NO:
 1. 4. Themutant 3-HBDH of claim 3, wherein the amino acid at the positioncorresponding to position 66 of SEQ ID NO: 1 is substituted with Tyr(66Tyr), or Asn (66Asn); position 109 of SEQ ID NO: 1 is substitutedwith Glu (109Glu); position 113 of SEQ ID NO: 1 is substituted with Thr(113Thr); position 125 of SEQ ID NO: 1 is substituted with Phe (125Phe);position 140 of SEQ ID NO: 1 is substituted with Val (140Val); position144 of SEQ ID NO: 1 is substituted with Arg (144Arg); position 145 ofSEQ ID NO: 1 is substituted with Gly (145Gly); position 195 of SEQ IDNO: 1 is substituted with Gln (195Gln) or Leu (195Leu); position 197 ofSEQ ID NO: 1 is substituted with Ile (197Ile); position 217 of SEQ IDNO: 1 is substituted with Val (217Val); position 223 of SEQ ID NO: 1 issubstituted with Val (223Val); position 232 of SEQ ID NO: 1 issubstituted with Cys (232Cys), Tyr (232Tyr), or Trp (232Trp); position239 of SEQ ID NO: 1 is substituted with Tyr (239Tyr), or Trp (239Trp);position 250 of SEQ ID NO: 1 is substituted with Met (250Met); and/orposition 257 of SEQ ID NO: 1 is substituted with Met (257Met) or Gln(257Gln).
 5. The mutant of claim 4, wherein the amino acid at theposition corresponding to position 37 of SEQ ID NO: 1 is substitutedwith Tyr (37Tyr); position 202 of SEQ ID NO: 1 is substituted with Gly(202Gly), and/or position 230 of SEQ ID NO: 1 is substituted with Leu(230Leu).
 6. The mutant 3-HBDH of claim 3, wherein the mutant 3-HBDH hasone or more mutation selected from: 125Phe; 144Arg; 232Cys; 250Met;230Leu and 250Met; 37Tyr and 250Met; 37Tyr, 230Leu and 250Met; 202Gly,230Leu and 250Met; 37Tyr, 145Gly, 230Leu and 250Met; and/or 37Tyr,195Gln, 230Leu and 250Met corresponding to positions of SEQ ID NO:1. 7.The mutant 3-HBDH of claim 1, wherein the mutant 3-HBDH comprises anamino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence of any of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 8.The mutant 3-HBDH of claim 1, wherein improved performance relative tothe wild-type 3-HBDH further comprises increased substrate affinity, for3-hydroxybutyrate, relative to the wild-type 3-HBDH; and/or increasedcofactor affinity, for NAD or a derivative thereof, wherein the derivateis carba-NAD, relative to the wild-type 3-HBDH.
 9. The mutant 3-HBDH ofclaim 8, wherein the mutant 3-HBDH has an at least 2-fold increasedstability relative to the wild-type 3-HBDH, an at least 3-fold increasedstability, an at least 4-fold increased stability, or an at least 5-foldincreased stability; and/or wherein the mutant 3-HBDH has an increasedsubstrate and/or cofactor affinity relative to the wild-type 3-HBDH, anincreased affinity for (i) 3-hydroxybutyrate and/or (ii) nicotinamideadenine dinucleotide (NAD) or a functionally active derivative thereofand/or wherein the substrate and/or cofactor affinity is increased by atleast 5%, at least 10%, at least 15% , or at least 20%.
 10. A nucleicacid encoding the mutant 3-HBDH of claim
 1. 11. A cell comprising themutant 3-HBDH of claim 1 or the nucleic acid of claim
 10. 12. A methodof determining the amount or concentration of 3-hydroxybutyrate in asample, the method comprising a) contacting the sample with the mutant3-HBDH of claim 1 under conditions conducive to the activity of the3-HBDH; b) reacting 3-hydroxybutyrate with nicotinamide adeninedinucleotide (NAD) or a functionally active derivative thereof; and c)determining the change in the redox state of NAD or the derivativethereof; thereby determining the amount or concentration of3-hydroxybutyrate in the sample.
 13. The method of claim 12, a) whereinthe determining of the change in the redox state of NAD or the derivatethereof includes the determination of the concentration of (i) NAD orthe derivate thereof and/or (ii) NADH or the derivate thereof; and/or b)wherein the determining the change in the redox state of NAD or thederivative thereof is electrochemically or optically; and/or c) whereinthe method further comprises determining the amount or concentration ofacetoacetate and/or acetone; and/or d) wherein the method furthercomprises determining the amount or concentration of glucose; and/or e)wherein the functionally active derivative of NAD is carba-NAD; and/orf) wherein the mutant 3-HBDH is part of a sensor, a test strip, a testelement, a test strip device or a liquid test; and/or g) wherein thesample is a body fluid.
 14. A device for determining the amount orconcentration of 3-hydroxybutyrate in a sample comprising the mutant3-HBDH according to claim 1 and a further component required for saiddetermining.
 15. The device according to claim 14, a) wherein the deviceis selected from the group consisting of a sensor or a test strip;and/or b) wherein the device further allows for determining the amountor concentration of glucose in the sample.
 16. The mutant of 3-HBDHaccording to claim 2, wherein the mutant 3-HBDH has only a mutationselected from: 250Met; 230Leu and 250Met; 37Tyr and 250Met; 37Tyr,230Leu and 250Met; 202Gly, 230Leu and 250Met; 37Tyr, 145Gly, 230Leu and250Met; or 37Tyr, 195Gln, 230Leu and 250Met corresponding to positionsof SEQ ID NO:
 1. 17. The mutant of 3-HBFB according to claim 7, whereinthe mutant 3-HBDH comprises a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 18. The mutant of 3-HBFBaccording to claim 7, wherein the mutant 3-HBDH consists of a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 19.The mutant 3-HBDH of claim 1, wherein the amino acid at the positioncorresponding to position 250 of SEQ ID NO: 1 is substituted with Met(250Met).
 20. The device according to claim 15, wherein the sensor is anelectrochemical sensor or an optical sensor.